Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, South China Agricultural University, Guangzhou 510642, China.
Engineering Research Center of Biocontrol, Ministry of Education, Guangzhou 510642, China.
Genes (Basel). 2020 Oct 10;11(10):1178. doi: 10.3390/genes11101178.
(Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in , the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for has yet been reported. Here, we selected seven housekeeping genes of and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using software, which contains four different programs (, Δ, , and ). Pairwise variation was analyzed by software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: and for the developmental stage; and for sex, and for different tissues (head, thorax, abdomen); and between two populations; and for different temperatures (5, 15, 25, 35 °C); and and for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP.
(沃特斯顿)是亚洲柑橘木虱(ACP)的主要寄生蜂，是中国南方柑橘领域中柑橘黄龙病的破坏性柑橘害虫和载体。为了研究目标基因在中的功能，在不同的实验条件下进行逆转录定量聚合酶链反应（RT-qPCR），筛选特定的参考基因是至关重要的。然而，目前还没有针对的参考基因。在这里，我们选择了的七个管家基因，并使用软件评估了它们在六个条件（发育阶段、性别、组织、种群、温度、饮食）下的稳定性，该软件包含四个不同的程序（、Δ、、和）。通过软件进行成对差异分析，以确定在 RT-qPCR 分析中最佳参考基因的数量。结果表明，不同的研究重点有更好的参考基因：和用于发育阶段；和用于性别，和用于不同的组织（头、胸、腹）；和在两个种群之间；和用于不同的温度（5、15、25、35°C）；和用于不同的喂养饮食。此外，当在不同的温度和组织处理中选择两个最佳和两个最不合适的参考基因时，相应的（作为报告基因）的表达模式有很大差异。我们的研究首次为 RT-qPCR 分析提供了更全面的参考基因列表，这对于研究该 ACP 天然敌害中的目标基因（s）的功能应该是有益的。
